A. phagocytophilum–induced actin phosphorylation selectively mediates I. scapularis salp16 gene promoter specificity. (A) Immunoblots showing the levels of phosphorylated actin, TBP, and RNAPII in A. phagocytophilum–infected mock (buffer alone) or ipak1-silenced tick nuclear extracts. Total actin served as the loading control. (B) EMSAs performed with the biotin-labeled salp16 or salp20 promoter TATA-binding regions and A. phagocytophilum–infected mock or ipak1-silenced nuclear extract proteins. Band shifts, salp16-, or salp20-free probes are indicated with arrows. Wedges indicate decreasing amounts of nuclear extracts (3, 2, and 1 µg). (C) Biotinylated DNAP with salp16 or salp20 probes and A. phagocytophilum–infected nuclear extract proteins. DNA precipitates were probed with actin-pTyr, anti-TBP, or anti-RNAP. Nuclear extracts were probed with anti-actin (input) as a loading control. Representative gel images from three independent experiments are shown in all panels.